Should We Perform Semen Analysis, DNA Fragmentation, and Hypo-osmotic Swelling Tests together?

Semen analysis, sperm DNA fragmentation (SDF) and hypo-osmotic swelling test (HOST) are usually performed for the evaluation of sperm fertilizing ability. There are some debates over the necessity of SDF and HOST incorporation in male infertility work-up.Semen of 77 men was evaluated by SDF and HOST through three semen analyses. Sperm parameters were arranged into different categories: <5%, 5-15%, >15% for normal morphology; <50%, 50-70%, >70 % for motility; and <10, 10-20, 21-34, 35-50, >50 million/ml for concentration. SDF analysis was performed and values under 30% were assumed to be normal. Normal range of HOST was considered to be >60%.Only normal sperm morphology had significant relationship with DF rate (P<0.001). Normal morphology, motility, and concentration of sperms had significant relationship with HOST (P<0.001, 0.05, and <0.003,respectively). There was a significant negative correlation between sperm morphology and DF rate. The correlations between sperm parameters and percentage of HOST were significantly positive (r: 0.44, 0.19, and 0.32 for morphology, motility, and concentration, respectively). Receiver operating characteristic curve (ROC) showed that sperm morphology is a strong predictor of the rate of DF and HOST (accuracy: 0.74' and 0.81, respectively). The best sperm morphology cut off point for DF and HOST rate prediction was 4.5% and 5.5%, respectively.Sperm morphology had significant correlation with DF rate and HOST and is supposed to be a predictor for these tests. Performing these three tests collectively for evaluation of semen samples would not be necessarily required in all cases.

Sperm DNA integrity for correct transmission of paternal genetic information (6) is the basis for SDF assay. This test is related to the presence of breaks in one or two strands of DNA in human spermatozoa (7,8). Defective chromatin condensation during spermiogenesis, apoptosis during spermatogenesis and oxidative stress (9) are major mechanisms leading to DNA damage. Although sperm DNA damage may be transmitted to the next generation, it has been reported that level of DNA fragmentation (DF) did not predict pregnancy outcome in intra cytoplasmic sperm injection (ICSI) cycles (10). Functional integrity of sperm membrane as a barrier between intra and extra cellular spaces and a sign for DNA integrity (11) can be assessed by hypo-osmotic swelling test (HOST). HOST is a common, low cost, simple and reliable test (12, 13) which may reveal functional ability of sperms including acrosome reaction, sperm capacitation, and the binding of spermatozoa to the oocyte surface (14). In viable spermatozoa, water (fluid) passes across the sperm membrane and causes swelling in the sperm tail (15). Different tail patterns may happen from (a) to (g) according to World Health Organization (WHO) where (a) is a dead spermatozoa and without tail changing. (b)-(g) have various types of tail changes. In case of as the nozoospermia and testicular immotile spermatozoa, HOST is useful for distinguishing of dead spermatozoa from viable immotile sperm (16,17).
There are dissimilarities in reports of relationship among routine semen analysis, SDF, and HOST. Negative correlation (18,19), and no correlation between sperm parameters and SDF (20,21) have been reported. Also, the reported correlations are different between sperm parameters and HOST values (22)(23)(24)(25)(26)(27). In Stanger's report, there was a strong correlation among sperm parameters, DF rate, and HOST value (28). In spite of these dissimilarities, existence of the correlations and predictive values among these three tests may lead to lack of necessity for performing these tests together in IVF labs. To the best of our knowledge, this is the first investigation which statistically analyzes the relationship of three functional sperm tests in the lab. In this case, we investigated the distribution of levels of DF and HOST values in different sperm parameter categories, the correlation between sperm parameters and DF, and sperm membrane response in hypo-osmolar condition.
Sperm parameters as predictors of DF rate and HOST were analyzed as well.

Patients and semen analysis
This cross-sectional study was conducted on 77 infertile couples (age range, 24-50 years) who were referred to IVF lab of Mehregan Hospital, Babol, Iran. Sample size was determined according to a similar article with confidence level of 95% and power of 80% (28). Ethics Committee center approved the study. Semen sample was collected with the aid of spouses after 3-5 days of abstinence.
After complete liquefaction, each sample was aliquoted for three separate portions. All samples were observed under light microscope. Sperm concentration and motility were assessed according to WHOguidelines (29). Sperm morphology was assessed according to Tygerberg strict criteria (30) after Papanicolaou staining procedure. A second aliquot of each sample was prepared immediately for DF rate assessment, and a third aliquot for HOST (12). Semen samples with ≥ 60% positive swelling tail reaction were considered as normal while those with less than 60% positive swelling tail reaction were considered as abnormal.

Statistical analyzes
Statistical analyses were performed by SPSS software (version 23, SPSS Inc, Chicago, IL, USA).
Categorical data were analyzed by chi-square test.
The cut off values were determined by ROC analysis and sensitivity and specificity were calculated. A p-value of less than 0.05 was considered as statistically significant.

Conventional semen analysis
The mean (±SD) of sperm concentration was  (Fig. 1). Half of the samples in two categories of sperm morphology i.e. <5% and 5-15%, had the DF rate above 30%. The distribution of normal HOST score in category of >15% normal morphology was higher in comparison to others (Fig. 1A).
There was a high significant negative correlation between sperm normal morphology and DF (r=-0.6; P≤ 0.001, Table 1). The category of>15%sperm with normal morphology had a lower DF than the other categories (Fig. 1A). The relationships between sperm motility and concentration with DF were not statistically significant (P= 0.1, P= 0.9, Table 1).Sperm parameters as predictors of DF were evaluated by ROC curve (Fig. 2).

Hypo-osmoticswelling test
The mean (±SD) of HOST score was 41.49 (± 19.37). Distribution of different patterns of the sperm tail response to hypo-osmotic stress according to WHO guidelines (29) are illustrated in  Table 2). Also, there was a negative relationship between SDF rate and percentage of HOST (r= -0.50; P<0.001). To evaluate the values of sperm parameters as predictors for HOST, the ROC curve was illustrated (Fig. 4). Diagnostic values and different cut off points of morphology, motility and concentration for prediction of SDF and HOST are shown in Table 3, 4. Among sperm parameters, morphology was the best predictor because it had an accuracy of 0.74 and 0.81 for DF rate and percentage of HOST, respectively (Fig. 2, 4).

Discussion
Routine semen analysis is the first line of sperm evaluation in most fertility clinics.
Limitations and insufficient value of this simple test lead to proposition of sperm DNA integrity (31) and HOST (32,33). There are some debates over the necessity of DF test incorporation in male infertility work-up (34,35). This study attempted to clarify the requirement of these functional tests routinely in IVF clinics.
The distribution of levels of DF in different sperm parameters categories were as follows; in half of samples with 5-15% normal morphology, DF was <30%, while in <5% category it reached up to 60%. The distribution of normal HOST value (≥60%) in category of >15% normal morphology was higher than the others (Fig. 1).
In the present study, there was a significant negative correlation between sperm morphology and DF rate. The correlation between sperm parameters and HOST was positive. There are some reports that indicate a negative correlation between sperm parameters and DF rate (18,19,36 In the present study concerning sperm DF and HOST, the best cut off points for sperm parameters were verified. We also confirmed which sperm parameter(s) is (are) valuable as predictor(s) of DF rate and normal range of HOST. According to our findings, morphology is the best predictor of sperm DF rate and HOST. In men whose sperm normal morphology is <5%, the DF≥30% and HOST< 60%would be expected. In a study, sperm morphology and motility were reported as main parameters for prediction of DF rate (36). In the present report, morphology is the best predictor not only for sperm DF rate but also for HOST.
In conclusion, there are relationships between DF and HOST values with sperm parameters.